This Project will investigate the relationships between hepatic cholesterol metabolism and atherosclerosis with the goal of defining the role of ACAT2 in atherosclerosis development. In the past funding period, we have identified two ACAT enzymes, ACAT1 and ACAT2, and have shown that ACAT2 is the form of the enzyme located in the hepatocyte and the enterocyte. In specific aim 1, we propose to determine the importance of ACAT2 in intestinal cholesterol absorption and the subsequent transport in chylomicrons. We will address the hypothesis that cholesterol esterification by ACAT2 in intestinal mucosal cells is required for bulk cholesterol incorporation and transport in chylomicrons, thereby determining the efficiency of cholesterol absorption. A corollary is that the absence of ACAT2 will limit the efficiency of cholesterol esterification and absorption thereby promoting cholesterol return to the intestinal lumen via the ABC G5/G8 transporter complex. Lymph duct cannulated ACAT2 KO mice crossed with ABC G5/G8 transgenic or KO mice will be studied for the extent of cholesterol and sitosterol absorption. In specific aim 2, we propose to ascertain the role of ACAT2 in the assembly and secretion of apoB-containing lipoproteins by the liver. We will address the hypothesis that ACAT2 increases the efficiency of hepatic apoB secretion and contributes cholesteryl esters to the core of these lipoproteins, in the process contributing to the increased atherogenicity observed when cholesteryl ester secretion is high. Isolated liver perfusions will be used to compare ACAT2-/-mice, ACAT1-/- mice and wildtype mice. In specific aim 3, we will test the hypothesis that oleoyI-CoA derived from SCD-1 is a primary source of oleoyl-CoA substrate available to ACAT2 and that blocking SCD-1 activity will limit the accumulation and hypersecretion of cholesteryl esters by the liver. A corollary to this hypothesis is that downregulation of SCD-1 by n-3 fatty acids will also limit ACAT2-derived choiesteryl ester accumulation. In specific aim 4, we will determine the role of ACAT2 in the development of atherosclerosis by examining the extent of atherosclerosis in mice with and without ACAT2. We will test the hypothesis that cholesteryl esters, synthesized by ACAT2 in the liver and secreted with apoB100-containing lipoproteins, contribute to increased LDL particle size and atherogenicity. ACAT2 deficient mice crossed into the LDL receptor deficient, apoB100-only mouse will be compared with ACAT1 deficient mice in the same genetic model. These studies will permit an assessment of the role of hepatic ACAT2 in promoting atherosclerosis.